During apoptosis, the cell employs mechanisms to achieve a rapid and tidy destruction of the nucleus and genomic DNA. The enzymes which carry out internucleosomal DNA fragmentation, chromatin condensation, and nuclear disassembly are present in a latent or inhibited form in healthy (nonapoptotic) cells. Previously we partially characterized a novel DNase activity appearing in the cytosol of cells initiating apoptosis, as well as a novel inhibitory activity of this DNase present in healthy cells, using a cell-free system. In 1998 other laboratories described a novel caspase-activated DNase (CAD or DNA Fragmentation Factor-40) and a caspase-cleavable specific inhibitor of it (ICAD or DNA Fragmentation Factor-45). To determine the relationship between these proteins and our activities, we prepared pure recombinant His6- tagged human ICAD and generated a high-quality antiserum against human (but not mouse) ICAD. We demonstrated that our apoptotic DNase and DNase-inhibitor activities correspond to CAD and ICAD, respectively. Our studies confirm and extend the characterization of a mechanism in which a constitutive endonuclease, CAD, binds an inhibitor, ICAD, present in excess in healthy cells, but when apoptosis is triggered, activated effector caspases cleave ICAD and thereby deinhibit CAD. We also demonstrated the presence of two ICAD forms (ICAD-L (45kDa) and ICAD-S (34 kDa)), probably arising by alternatively mRNA splicing, in healthy human cells, namely HeLa, Jurkat lymphoma, SK-N-MC neuroblastoma, and WI-38 fibroblast cells. We have begun studies on ICAD and CAD gene expression in experimentally amenable mouse tissues and cell lines. By RT-PCR, we cloned mouse ICAD and mouse and human CAD cDNAs. Then we expressed recombinant His6-tagged mouse ICAD protein in E. coli and purified it. Human and mouse CAD proteins were also well expressed in E. coli but were insoluble. We generated an antiserum to mouse ICAD which recognizes mouse and human ICAD-L and ICAD-S. By Western blot analyses, we demonstrated that ICAD-L and -S are expressed in all mouse tissues examined, but their steady state levels and L/S ratios are markedly varied. In spleen ICAD-L is very abundant and predominates over ICAD-S, while in brain and heart ICAD-S is moderately abundant and predominates over ICAD-L. Both forms are present in very low amounts in liver, lung, and kidney. In mouse cell lines, both ICAD forms are usually expressed but in highly variable amounts; for example, two neuroblastoma lines (NS20Y and N1E115) exhibited very high levels of ICAD-L, while NIH3T3 fibroblasts had low levels of both forms. We conclude that although ICAD may be a constitutively expressed gene, there are marked cell-type specific variations in its expression and mRNA splicing. These results indicate that the ICAD gene is strongly regulated by variables which remain to be identified. - Apoptosis, programmed cell death, DNase, DNA fragmentation factor, CAD, ICAD, DFF, caspase, nucleus